At entheses, tendons and bones are bridged by mineralized fibrocartilage, joined to tissues through dedicated interfaces. Tendons and bones are characterized by cells interconnected thanks to their underlying dense networks. Nanotubes connect tenocytes in tendons, allowing cellular crosstalk and providing biomechanical stability. Osteocytes are involved in bone mechanoresponsiveness and mineralization: they are encased into cavities and their cellular processes run through channels, forming the osteocyte lacunocanalicular network. Here, we explore the structural connectivity between fibrocartilage and bone, exploiting rat enthesis as model system and focusing on two specific regions: the Achilles tendon insertion into calcaneus and the periosteal fibrocartilage, facilitating tendon sliding. Those regions are used to characterize the impact of loading environment on tissue connectivity. Central to our approach is rhodamine staining, employed to trace connections between tissues. This information is interpreted using data on tissue microstructure, organization and composition, acquired combining high-resolution imaging methods. At the enthesis, we observe potential connections between trabecular bone marrow and mineralized fibrocartilage through a subchondral channel network perforating the interface. Direct cellular connections between bone and fibrocartilage cells are rare: canaliculi mostly stop or switch direction at the cement line. Yet, we observed a high density of canaliculi around perforating channels, which reach fibrochondrocyte lacunae. Such connections seem practically absent at the periosteal region. Our findings are preliminary but suggest that inter-tissue connectivity is required to support the enthesis load-bearing function. To understand multi-tissue biochemical cellular crosstalk, the physical infrastructure enabling this communication is also a critical feature to investigate.

The functional integration of soft tissues into bone requires complex adaptation strategies [1]. Tendon and bone are both characterized by cells (tenocytes and osteocytes) that communicate extensively thanks to their dense networks: nanotubes in tendons and the osteocyte lacunocanalicular network (LCN) in bone. These networks serve multiple functions: nanotubes in tendon are believed to provide biomechanical stability [2] while the LCN is involved in bone mechanoresponsiveness [3] and mineralization [4]. At the interface between theses tissues lies the enthesis, a specialized insertion site often featuring fibrocartilage (FC), a fibrous form of cartilage reinforced with minerals before anchoring to bone. Our research focuses specifically on the Achilles tendon insertion, comparing enthesis with periosteal FC, two contiguous tissues sustaining different loading conditions: tension at enthesis and compression/shear at periosteal FC. We have previously revealed microstructural [5] and material heterogeneity [6] of mineralized FC. The aim of this study is to investigate the behavior of fibrochondrocytes (FC cells) and their physical interaction pathways with bone functional porosity. Rat samples were stained with rhodamine and then micro-computed tomography, quantitative backscattered electron imaging, second harmonic generation imaging and confocal laser scanning microscopy (CLSM) were combined on the same locations to highlight functional porosity at multiple length scales, as well as its link with mineral content and matrix organization. At the enthesis, we identified a connection path between trabecular bone marrow space and FC through the anisotropic subchondral channel network, perforating the bone-FC interface at several locations. Communication between bone and FC cells seems to be primarily indirect: osteocytes are highly connected to perforating channels through many canaliculi, these channels crossing the bone-FC interface and reaching the fibrochondrocyte rows. The striking absence of such connections at the adjacent periosteal region suggests that communication between bone and fibrocartilage may be needed for a proper enthesis biomechanical function.

Background: Abdominal aortic aneurysm (AAA) is a chronic inflammatory disease that poses several challenges. Given the increasing evidence that AAA patients are more likely to develop cancer and the importance of its early detection, we strived to develop a non-invasive tool based on serial FDG-PET/CT scan examinations to identify, among AAA patients, those at risk of cancer. Methods: Between 2006 and 2011 we recruited 149 AAA patients, free of cancer at baseline, and followed them until the end of 2021. All patients underwent an FDG-PET/CT scan at inclusion and possibly more scans during follow-up. At each medical imaging examination, the aneurysmal FDG uptake was recorded. Patients were stratified based on their aortic wall PET status (negative/positive). Any occurrence of cancer was reported. A Cox regression analysis and competing-risk modeling were applied to the data. Results: The proportion of AAA patients who developed cancer was 31.5% (mean time to diagnosis was 5.7 ± 3.4 years) and the death rate was 59%. A difference in cancer incidence between PET+ and PET− patients was detected (46.8% vs. 27.3%; HR = 1.96, 95%CI: 1.07–3.57, p = 0.028). Moreover, AAA patients undergoing surgical treatment had a lower risk of cancer than unoperated patients (28% vs. 50%; HR = 0.41, 95%CI: 0.21–0.80, p = 0.009). Conclusions: In AAA patients, diagnostic imaging with an FDG-PET/CT scan can help identify those patients at a higher risk of developing cancer. Moreover, the higher cancer risk in non-surgically treated patients calls for further analysis of associations between aneurysm growth and malignant disease.

A demanding task of the musculoskeletal system is the attachment of tendon to bone at entheses. This region often presents a thin layer of fibrocartilage (FC), mineralized close to the bone and unmineralized close to the tendon. Mineralized FC deserves increased attention, owing to its crucial anchoring task and involvement in enthesis pathologies. Here, we analyzed mineralized FC and subchondral bone at the Achilles tendon-bone insertion of rats. This location features enthesis FC anchoring tendon to bone and sustaining tensile loads, and periosteal FC facilitating bone-tendon sliding with accompanying compressive and shear forces. Using a correlative multimodal investigation, we evaluated potential specificities in mineral content, fiber organization and mechanical properties of enthesis and periosteal FC. Both tissues had a lower degree of mineralization than subchondral bone, yet used the available mineral very efficiently: for the same local mineral content, they had higher stiffness and hardness than bone. We found that enthesis FC was characterized by highly aligned mineralized collagen fibers even far away from the attachment region, whereas periosteal FC had a rich variety of fiber arrangements. Except for an initial steep spatial gradient between unmineralized and mineralized FC, local mechanical properties were surprisingly uniform inside enthesis FC while a modulation in stiffness, independent from mineral content, was observed in periosteal FC. We interpreted these different structure-property relationships as a demonstration of the high versatility of FC, providing high strength at the insertion (to resist tensile loading) and a gradual compliance at the periosteal surface (to resist contact stresses). STATEMENT OF SIGNIFICANCE: Mineralized fibrocartilage (FC) at entheses facilitates the integration of tendon in bone, two strongly dissimilar tissues. We focus on the structure-function relationships of two types of mineralized FC, enthesis and periosteal, which have clearly distinct mechanical demands. By investigating them with multiple high-resolution methods in a correlative manner, we demonstrate differences in fiber architecture and mechanical properties between the two tissues, indicative of their mechanical roles. Our results are relevant both from a medical viewpoint, targeting a clinically relevant location, as well as from a material science perspective, identifying FC as high-performance versatile composite.

AimsDietary cholesterol and palmitic acid are risk factors for cardiovascular diseases (CVDs) affecting the arteries and the heart valves. The ionizing radiation that is frequently used as an anticancer treatment promotes CVD. The specific pathophysiology of these distinct disease manifestations is poorly understood. We, therefore, studied the biological effects of these dietary lipids and their cardiac irradiation on the arteries and the heart valves in the rabbit models of CVD.Methods and ResultsCholesterol-enriched diet led to the thickening of the aortic wall and the aortic valve leaflets, immune cell infiltration in the aorta, mitral and aortic valves, as well as aortic valve calcification. Numerous cells expressing α-smooth muscle actin were detected in both the mitral and aortic valves. Lard-enriched diet induced massive aorta and aortic valve calcification, with no detectable immune cell infiltration. The addition of cardiac irradiation to the cholesterol diet yielded more calcification and more immune cell infiltrates in the atheroma and the aortic valve than cholesterol alone. RNA sequencing (RNAseq) analyses of aorta and heart valves revealed that a cholesterol-enriched diet mainly triggered inflammation-related biological processes in the aorta, aortic and mitral valves, which was further enhanced by cardiac irradiation. Lard-enriched diet rather affected calcification- and muscle-related processes in the aorta and aortic valve, respectively. Neutrophil count and systemic levels of platelet factor 4 and ent-8-iso-15(S)-PGF2α were identified as early biomarkers of cholesterol-induced tissue alterations, while cardiac irradiation resulted in elevated levels of circulating nucleosomes.ConclusionDietary cholesterol, palmitic acid, and cardiac irradiation combined with a cholesterol-rich diet led to the development of distinct vascular and valvular lesions and changes in the circulating biomarkers. Hence, our study highlights unprecedented specificities related to common risk factors that underlie CVD.

Graft-versus-host disease (GVHD) is a major cause of toxicity after allogeneic hematopoietic cell transplantation (allo-HCT). While rapamycin (RAPA) is commonly used in GVHD prophylaxis in combination with a calcineurin inhibitor (CNI), the understanding of its mechanism of action on human T cells is still incomplete. Here, we performed an extensive analysis of RAPA effects on human T cells in a humanized mouse model of GVHD, in ex - vivo T cell cultures and in patients given RAPA plus tacrolimus as GVHD prophylaxis after nonmyeloablative allo-HCT. We demonstrate that RAPA mitigates GVHD by decreasing T cell engraftment and differentiation, inhibiting CD8 + T cell activation and increasing the long-term IL-2 secretion, thereby supporting regulatory T cell (Treg) proliferation. In contrast, graft-versus-leukemia effects were not abrogated, as RAPA-treated T cells had increased resistance to apoptosis and retained their effector function and proliferative capacity upon re-stimulation. Importantly, we found that RAPA impact on Treg and CD8 + T cells was closely dependent upon IL-2 signaling and that therapeutic options interfering with IL-2, such as calcineurin inhibitors, antagonize the IL-2-dependent promotion of Treg mediated by RAPA. Our results suggest that RAPA immunological efficacy could be improved in combination with drugs having possible synergistic effects such as the hypomethylating agent 5-azacytidine.

The enthesis allows the insertion of tendon into bone thanks to several remarkable strategies. This complex and clinically relevant location often features a thin layer of fibrocartilage sandwiched between tendon and bone to cope with a highly heterogeneous mechanical environment. The main purpose of this study was to investigate whether mineralized fibrocartilage and bone close to the enthesis show distinctive three-dimensional microstructural features, possibly to enable load transfer from tendon to bone. As a model, the Achilles tendon-calcaneus bone system of adult rats was investigated with histology, backscattered electron imaging and micro-computed tomography. The microstructural porosity of bone and mineralized fibrocartilage in different locations including enthesis fibrocartilage, periosteal fibrocartilage and bone away from the enthesis was characterized. We showed that calcaneus bone presents a dedicated protrusion of low porosity where the tendon inserts. A spatially resolved analysis of the trabecular network suggests that such protrusion may promote force flow from the tendon to the plantar ligament, while partially relieving the trabecular bone from such a task. Focusing on the tuberosity, highly specific microstructural aspects were highlighted. Firstly, the interface between mineralized and unmineralized fibrocartilage showed the highest roughness at the tuberosity, possibly to increase failure resistance of a region carrying large stresses. Secondly, fibrochondrocyte lacunae inside mineralized fibrocartilage, in analogy with osteocyte lacunae in bone, had a predominant alignment at the enthesis and a rather random organization away from it. Finally, the network of subchondral channels inside the tuberosity was highly anisotropic when compared to contiguous regions. This dual anisotropy of subchondral channels and cell lacunae at the insertion may reflect the alignment of the underlying collagen network. Our findings suggest that the microstructure of fibrocartilage may be linked with the loading environment. Future studies should characterize those microstructural aspects in aged and or diseased conditions to elucidate the poorly understood role of bone and fibrocartilage in enthesis-related pathologies.

Platelet-rich plasma (PRP) is increasingly used in the treatment of musculoskeletal diseases. Its preservation by freezing it for the realization of multiple injections in clinical use has never been discussed. Calcaneal tendons of rats were surgically sectioned. Platelet concentration of the PRP was 2.5 x 106/µl with autologous plasma of rats. Frozen-thawed PRP was prepared by performing two cycles of freezing and thawing on PRP aliquots. Both platelet preparations were injected in the lesion. Biomechanical and histological evaluations were carried out after 7, 20 or 40 days post surgery. After 7 and 40 days, no significant difference was observed between the PRP and the frozen-thawed PRP group. There is however a difference 20 days after surgery: the ultimate tensile strength (UTS) was greater in the fresh PRP group. No obvious difference with histological aspect was observed between the two groups. In conclusion, fresh PRP and frozen-thawed PRP injections can lead to similar results in the healing process of section calcaneal tendons of rats. Improvements with fresh PRP are slight. PRP could thus be frozen to be preserved if multiple injections are needed (e.g. osteoarthritis).

Chemically-induced diabetic animal models have been employed in many areas of diabetes mellitus (DM) research, but managing post-induction animal survival rates remains one of the main downsides.The aim of the present study was to propose a reliable approach to animal management and monitoring after DM induction in a rabbit model in order to reduce animal mortality rates.DM was induced by injecting alloxan in 12 New Zealand White rabbits. A preventive subcutaneous glucose administration to counteract a potentially lethal hypoglycemic phase following alloxan injection was performed on individual bases. Blood glucose level (BGL) was checked hourly for the first 36 h, then every 2 h until the hyperglycemic state was confirmed.All 12 rabbits survived a 48-hour post-induction phase. The critical hypoglycemic phase's start points and duration differed significantly among the rabbits, lasting from 6.7 to 37 h (19.75 ± 8.44). The rabbits entered the final hyperglycemic phase 18 h at the earliest and 42 h at the latest after induction (26.63 ± 7.07). The average daily BGLs throughout the study period ranged from 268 to 512 mg/dL (413.73 ± 76.69). Eleven rabbits survived until the end of the experiment.The variability of rabbits' responses to alloxan injection emphasizes the importance of monitoring rabbit behavior and thoroughly checking BGLs, followed by a preventive glucose administration based on rabbits' individual needs for up to 36 h after alloxan injection. The proposed approach seems to reduce animal mortality.

Mean roughness (root mean square P q ): 65% higher at the interface between unmineralized (UFC) and mineralized

Background: Some studies focusing on the endovascular repair of abdominal aortic aneurysms (AAA) have demonstrated that cancer is often found in such patients. Retrospective investigations have also indicated that AAA patients have an increased risk of cancer. However, the incidence rate of cancer and survival rate in patients with AAA compared to the population remains unknown. Methods: We conducted a prospective, single center, non-randomized, comparative cohort study including 223 AAA patients and 1000 Coronary artery disease (CAD) patients enrolled in our department between 01/01/2008 and 31/12/2011 with annual follow-up until 31/12/2018. Based on statistics from the Belgian cancer registry Belgium, the incidence rate of cancer was calculated for both men and women in the general population. The primary endpoint was the cumulative incidence rate of cancer in all three groups and the secondary endpoint was overall survival. Results: After nine years of follow-up, the cancer incidence was 30·1% (95%CI: 21·6-39·3%) in AAA patients and 23·5% (95%CI: 20·0-27·1%) in CAD subjects, markedly higher than the Walloon population (17·4%). Moreover, the development of metastases among patients with AAA was higher compared to CAD patients (38·7% vs.18·6%, P=0·03). In terms of overall survival, the proportion of cancer deaths in AAA patients was twice as high as in CAD patients. Conclusions: This study confirms an increased incidence of cancers and decreased survival in AAA patients compared to the general population. Funding Statement: This work was supported by the European Program FP7 “Fighting aneurysmal diseases” (no. 200647). Audrey Courtois and Jacobine Munten were funded by the Aneurysmal Pathology Foundation. Declaration of Interests: The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. Ethics Approval Statement: The study was designed and conducted within the framework of the European program “Fighting Aneurysmal Disease (FAD; B70720095774 and B70720095773 9)” approved by the ethics committee (n°200647). All the participants were instructed on the study objectives and provided informed consent. The study was approved by the ethics board of the University hospital of Liege.

Background: Abdominal aortic aneurysm (AAA) is one of the leading causes of death in western countries. Surgery is still, at the present time, the sole treatment that has however a significant mortality and cost rate. Many pharmacological agents are under investigation aiming to reduce growth and prevent AAA rupture. These drugs target different pathological pathways and, notably, the excessive production of prostanoids by cyclooxygenases (COX). Intra-aneurysmal thrombus plays an adverse key role in the progression of AAA, platelets being a primary source of prostanoids as thromboxane A2.Objective: In this review, we summarize studies targeting prostanoids production and down-stream pathways in cardiovascular diseases, and more specifically in AAA.Results and Conclusion: Various inhibitors of COX or antagonists of prostanoids receptors have been investigated in AAA animal models with conflicting results. In human AAA, only a few number of studies focused on anti-platelet therapy mostly using acetylsalicylic acid (aspirin, ASA), a COX1 inhibitor. Finally, we report preliminary promising results of a model of AAA in rats receiving a thromboxane A2 inhibitor, BM-573 that induced a reduction of aneurysmal growth.

This article describes the experimental procedures used to observe if PRP can positively affect tendon healing. There are 4 main steps to follow: induce a lesion in the Achilles tendon; prepare PRP and inject it (or the saline solution); remove the tendon; and perform biomechanical, molecular, and histological evaluations. At each step, all the procedures and methods are described in detail, so they can be reproduced easily. Achilles tendons have been surgically sectioned (removal of a 5-mm long section). Afterwards, PRP or saline solution was injected to study whether PRP has a positive effect on the healing of the tendon. Three groups of 40 animals (a total of 120 rats were used in this study) were subdivided into 2 subgroups: PRP injection group and a saline injection control group. Rats were sacrificed at increasing time points (Group A: 5 days; Group B: 15 days; Group C: 30 days) and tendons were removed. 90 tendons underwent biomechanical testing before performing transcriptomic analysis and the 30 remaining tendons were submitted to histological analysis.